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A 4-year-old female Maltese dog was referred to our veterinary hospital with uveitis and conjunctivitis of the right eye. An ophthalmological evaluation revealed an intraocular mass that appeared to originate from the anterior uvea. Metastasis and regional invasion were not detected with CT examination. Enucleation of the right eye was recommended; however, the owner declined treatment. Six months later, the dog was re-presented with a right facial mass. At presentation, superficial lymph node enlargement was not appreciated, and no apparent alterations were noted on blood analysis or urinalysis. Computed tomography revealed an intraocular mass that invaded the surrounding tissues, including the frontal sinus. Presumed solitary ocular lymphoma with a large B-cell phenotype and Mott cell change was diagnosed via histopathological and immunohistochemical examination of a biopsy of the lesion. As the mass was too large for complete excision, neoadjuvant chemotherapy was administered. Complete remission was achieved using the L-COAP protocol and successful exenteration of the right eye. However, the dog was returned with enlargement of the right retropharyngeal lymph nodes. To the best of our knowledge, this is the first case report of presumed solitary ocular lymphoma with a large B-cell phenotype displaying Mott cell change in a dog. Key clinical message: This is the first reported case of a presumed solitary ocular lymphoma with a large B-cell phenotype and Mott cell change. Although systemic involvement was observed 6 mo after the initial visit, neoadjuvant chemotherapy and exenteration were effective., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

A 6-year-old castrated male mixed dog presented with a rapidly growing mass at the right chest wall two weeks after initial detection. A mesenchymal origin of the malignancy was suspected based on fine-needle aspiration. Computed tomography (CT) revealed that the mass originated from the right chest wall and protruded externally (6.74 × 5.51 × 4.13 cm 3 ) and internally (1.82 × 1.69 × 1.50 cm 3 ). The patient revisited the hospital because of breathing difficulties. Radiography confirmed pleural effusion, and ultrasonography-guided thoracocentesis was performed. The effusion was hemorrhagic, and microscopic evaluation showed no malignant cells. Before surgery, CT without anesthesia was performed to evaluate the status of the patient. The 7-10th ribs were en bloc resected at a 3-cm margin dorsally and ventrally, and two ribs cranially and caudally from the mass. After recovering the collapsed right middle lobe of the lung due to compression from the internal mass with positive-pressure ventilation, a 3D-printed bone model contoured titanium mesh was tied to each covering rib and surrounding muscles using 2-0 blue nylon and closed routinely. The thoracic cavity was successfully reconstructed, and no flail chest was observed. The patient was histo-pathologically diagnosed with extraskeletal osteosarcoma. A CT scan performed 8 months after surgery showed no evident recurrence, metastasis, or implant failure. This is the first case report of chest wall reconstruction using titanium mesh in a dog. The use of a titanium mesh allows for the reconstruction of extensive chest wall defects, regardless of location, without major postoperative complications.

Given the previous SARS-CoV-2 pandemic and the inherent unpredictability of viral antigenic drift and shift, preemptive development of diverse neutralizing antibodies targeting a broad spectrum of epitopes is essential to ensure immediate therapeutic and prophylactic interventions during emerging outbreaks. In this study, we present a monoclonal antibody engineered for cross-reactivity to both wild-type and Delta RBDs, which, surprisingly, demonstrates enhanced neutralizing activity against the Omicron variant despite a significant number of mutations. Using an Escherichia coli inner membrane display of a human naïve antibody library, we identified antibodies specific to the wild-type SARS-CoV-2 receptor binding domain (RBD). Subsequent directed evolution via yeast surface display yielded JS18.1, an antibody with high binding affinity for both the Delta and Kappa RBDs, as well as enhanced binding to other RBDs (wild-type, Alpha, Beta, Gamma, Kappa, and Mu). Notably, JS18.1 (engineered for wild-type and Delta RBDs) exhibits enhanced neutralizing capability against the Omicron variant and binds to RBDs noncompetitively with ACE2, distinguishing it from other previously reported antibodies. This underscores the potential of pre-existing antibodies to neutralize emerging SARS-CoV-2 strains and offers insights into strategies to combat emerging viruses.

Laboratory automation with robot-assisted processes enhances synthetic biology, but its economic impact on projects is uncertain. We have proposed an experiment price index (EPI) for a quantitative comparison of factors in time, cost, and sample numbers, helping measure the efficiency of laboratory automation in synthetic biology and biomolecular engineering., Competing Interests: Declaration of interests H.M.W. declares no competing financial interest. J.K. has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Biomia, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Severe fever with thrombocytopenia syndrome virus (SFTSV) or Dabie bandavirus is an emerging pathogen responsible for SFTS. It is considered a novel threat to human health, given the high associated fatality. SFTSV is a segmented negative-strand RNA virus containing three single-stranded RNAs, with the M segment encoding the glycoproteins Gn and Gc. Gc is vital for viral entry into the host cell surface, along with the Gn protein. As the Gc is the surface-exposable antigen from virions, it is a critical diagnostic marker of infection. Although various SFTSV Gn or N protein-based sero-diagnostic methods have been developed, there are no commercially available sero-diagnostic kits. Therefore, we generated monoclonal antibodies (mAbs) against SFTSV Gc and explored their application in serum diagnostic tests to develop sensitive serodiagnostic tools covering broad-range genotypes (A to F). First, 10 SFTSV Gc antibody-binding fragments (Fabs) were isolated using a phage display system and converted into human IgGs. Enzyme-linked immunosorbent assays (ELISA) of the SFTSV and Rift Valley fever virus (RVFV: same genus as SFTSV) Gc antigens showed that all antibodies attached to the SFTSV Gc protein had high affinity. An immunofluorescence assay (IFA), to verify the cross-reactivity of seven antibodies with high affinities for various SFTSV genotypes (A, B2, B3, D, and F) and detect mAb binding with intact Gc proteins, revealed that five IgG type mAbs were bound to intact Gc proteins of various genotypes. Six high-affinity antibodies were selected using ELISA and IFA. The binding capacity of the six antibodies against the SFTSV Gc antigen was measured using surface plasmon resonance. All antibodies had high binding capacity. Consequently, these antibodies serve as valuable markers in the serological diagnosis of SFTSV., Competing Interests: We declare competing interests related to the patent titled “Monoclonal antibodies for Gc protein of Severe Fever Thrombocytopenia Syndrome Virus, and use thereof” identified by patent number No. 10-2021-0163,224 (Korean patent application)., (© 2024 Published by Elsevier B.V.)

Background: Diabetic retinopathy (DR) is a vision-threatening complication that affects virtually all diabetic patients. Various treatments have been attempted, but they have many side effects and limitations. Alternatively, stem cell therapy is being actively researched, but it faces challenges due to a low cell survival rate. In this study, stem cells were pretreated with sirolimus, which is known to promote cell differentiation and enhance the survival rate. Additionally, the subconjunctival route was employed to reduce complications following intravitreal injections., Methods: Diabetes mellitus was induced by intraperitoneal injection of 55 mg/kg of streptozotocin (STZ), and DR was confirmed at 10 weeks after DM induction through electroretinogram (ERG). The rats were divided into four groups: intact control group (INT), diabetic retinopathy group (DR), DR group with subconjunctival MSC injection (DR-MSC), and DR group with subconjunctival sirolimus-pretreated MSC injection (DR-MSC-S). The effects of transplantation were evaluated using ERG and histological examinations., Results: The ERG results showed that the DR-MSC-S group did not significantly differ from the INT in b-wave amplitude and exhibited significantly higher values than the DR-MSC and DR groups ( p < 0.01). The flicker amplitude results showed that the DR-MSC and DR-MSC-S groups had significantly higher values than the DR group ( p < 0.01). Histological examination revealed that the retinal layers were thinner in the DR-induced groups compared to the INT group, with the DR-MSC-S group showing the thickest retinal layers among them., Conclusions: Subconjunctival injection of sirolimus-pretreated MSCs can enhance retinal function and mitigate histological changes in the STZ-induced DR rat model.

Bacterial small RNAs (sRNAs) function in post-transcriptional regulatory responses to environmental changes. However, the lack of eukaryotic RNA interference-like machinery in bacteria has limited the systematic engineering of RNA repression. Here, we report the development of clustered regularly interspaced short palindromic repeats (CRISPR)-guided dead CRIPSR-associated protein 13a (dCas13a) ribonucleoprotein that utilizes programmable CRISPR RNAs (crRNAs) to repress trans-acting and cis-acting sRNA as the target, altering regulatory mechanisms and stress-related phenotypes. In addition, we implemented a modular loop engineering of the crRNA to promote modular repression of the target gene with 92% knockdown efficiency and a single base-pair mismatch specificity. With the engineered crRNAs, we achieved targetable single-gene repression in the polycistronic operon. For metabolic application, 102 crRNAs were constructed in the biofoundry and used for screening novel knockdown sRNA targets to improve lycopene (colored antioxidant) production in Escherichia coli. The CRISPR-dCas13a system will assist as a valuable systematic tool for the discovery of novel sRNAs and the fine-tuning of bacterial RNA repression in both scientific and industrial applications., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The need for efficient computational screening of molecular candidates that possess desired properties frequently arises in various scientific and engineering problems, including drug discovery and materials design. However, the enormous search space containing the candidates and the substantial computational cost of high-fidelity property prediction models make screening practically challenging. In this work, we propose a general framework for constructing and optimizing a high-throughput virtual screening (HTVS) pipeline that consists of multi-fidelity models. The central idea is to optimally allocate the computational resources to models with varying costs and accuracy to optimize the return on computational investment. Based on both simulated and real-world data, we demonstrate that the proposed optimal HTVS framework can significantly accelerate virtual screening without any degradation in terms of accuracy. Furthermore, it enables an adaptive operational strategy for HTVS, where one can trade accuracy for efficiency., Competing Interests: B.-J.Y. is a member of the Advisory Board of Patterns., (© 2023 The Author(s).)

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Objectives: This study investigated the prevalence of feline chronic gingivostomatitis in urban feral cats in South Korea and analysed its risk factors., Methods: Three hundred and forty-five feral cats that visited the hospital for neutering using a trap-neuter-return approach were screened for feline chronic gingivostomatitis based on clinical criteria. In addition, we determined if body weight, sex and the presence of tongue lesions are risk factors for feline chronic gingivostomatitis. The difference in severity due to the presence or absence of risk factors, and the relationship between gross findings and histopathological lesions, were analysed by grading lesion severity., Results: Feline chronic gingivostomatitis was diagnosed in 92 cats. Disease prevalence did not significantly differ with body weight and sex but was significantly related to tongue lesions., Conclusions and Relevance: The prevalence of feline chronic gingivostomatitis in urban feral cats in South Korea was 26.6%. It was significantly more prevalent in cats that had tongue lesions. Severity was also significantly associated with tongue lesions. Feline chronic gingivostomatitis may be associated with an infectious agent that causes tongue lesions.

Genome editing technology is a powerful tool for programming microbial cell factories. However, rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of the bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we demonstrate the genome engineering of Corynebacterium glutamicum as a GC-rich model industrial bacterium by generating premature termination codons (PTCs) in desired genes using high-fidelity cytosine base editors (CBEs). Through this CBE-STOP approach of introducing specific cytosine conversions, we constructed several single-gene-inactivated strains for three genes ( ldh , idsA , and pyc ) with high base editing efficiencies of average 95.6% ( n  = 45, C6 position) and the highest success rate of up to 100% for PTCs and ultimately developed a strain with five genes ( ldh , actA , ackA , pqo , and pta ) that were inactivated sequentially for enhancing succinate production. Although these mutant strains showed the desired phenotypes, whole-genome sequencing (WGS) data revealed that genome-wide point mutations occurred in each strain and further accumulated according to the duration of CBE plasmids. To lower the undesirable mutations, high-fidelity CBEs (pCoryne-YE1-BE3 and pCoryne-BE3-R132E) was employed for single or multiplexed genome editing in C. glutamicum, resulting in drastically reduced sgRNA-independent off-targets. Thus, we provide a CRISPR-assisted bacterial genome engineering tool with an average high efficiency of 90.5% ( n  = 76, C5 or C6 position) at the desired targets. IMPORTANCE Rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies of bacterial CBEs have been developed, a risk of unidentified off-targets impeded genome editing for microbial cell factories. To address the issues, we identified the DNA off-targets for single and multiple genome engineering of the industrial bacterium Corynebacterium glutamicum using whole-genome sequencing. Further, we developed the high-fidelity (HF)-CBE with significantly reduced off-targets with comparable efficiency and precision. We believe that our DNA off-target analysis and the HF-CBE can promote CRISPR-assisted genome engineering over conventional gene manipulation tools by providing a markerless genetic tool without need for a foreign DNA donor.

As global interest in renewable energy continues to increase, there has been a pressing need for developing novel energy storage devices based on organic electrode materials that can overcome the shortcomings of the current lithium-ion batteries. One critical challenge for this quest is to find materials whose redox potential (RP) meets specific design targets. In this study, we propose a computational framework for addressing this challenge through the effective design and optimal operation of a high-throughput virtual screening (HTVS) pipeline that enables rapid screening of organic materials that satisfy the desired criteria. Starting from a high-fidelity model for estimating the RP of a given material, we show how a set of surrogate models with different accuracy and complexity may be designed to construct a highly accurate and efficient HTVS pipeline. We demonstrate that the proposed HTVS pipeline construction and operation strategies substantially enhance the overall screening throughput., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant morbidity and mortality worldwide. Despite a successful vaccination programme, the emergence of mutated variants that can escape current levels of immunity mean infections continue. Herein, we report the development of CT-P63, a broad-spectrum neutralizing monoclonal antibody. In vitro studies demonstrated potent neutralizing activity against the most prevalent variants, including Delta and the BA.1 and BA.2 sub-lineages of Omicron. In a transgenic mouse model, prophylactic CT-P63 significantly reduced wild-type viral titres in the respiratory tract and CT-P63 treatment proved efficacious against infection with Beta, Delta, and Omicron variants of SARS-CoV-2 with no detectable infectious virus in the lungs of treated animals. A randomized, double-blind, parallel-group, placebo-controlled, Phase I, single ascending dose study in healthy volunteers (NCT05017168) confirmed the safety, tolerability, and pharmacokinetics of CT-P63. Twenty-four participants were randomized and received the planned dose of CT-P63 or placebo. The safety and tolerability of CT-P63 were evaluated as primary objectives. Eight participants (33.3%) experienced a treatment-emergent adverse event (TEAE), including one grade ≥3 (blood creatine phosphokinase increased). There were no deaths, treatment-emergent serious adverse events, TEAEs of special interest, or TEAEs leading to study drug discontinuation in the CT-P63 groups. Serum CT-P63 concentrations rapidly peaked before declining in a biphasic manner and systemic exposure was dose proportional. Overall, CT-P63 was clinically safe and showed broad-spectrum neutralizing activity against SARS-CoV-2 variants in vitro and in vivo .

Corynebacterium glutamicum is widely used for a large-scale industrial producer of feed additive amino acids, such as l-lysine. Moreover, C. glutamicum has been engineered for producing various non-native chemicals, including terpenes. For the first time, C. glutamicum was engineered for co-production of l-lysine and heterologous squalene. To control metabolic fluxes for either the l-lysine biosynthesis pathway or the squalene biosynthesis pathway, pyruvate, an intermediate in the central metabolism, a node was regulated by a clustered regularly interspaced short palindromic repeat (CRISPR) interference system. Repressing pyc encoding for pyruvate carboxylase in the l-lysine producer (DM1919) and its derivatives resulted in 99.24 ± 7.63 mg/L total squalene and 6.25 ± 0.20 g/L extracellular lysine at 120 h. Furthermore, various oil overlays were tested for efficient co-productions. In situ extraction with corn oil (10%, v/v) exhibited a separation of 99.75% (w/v) of total squalene (intra- and extracellular squalene), while l-lysine can be secreted in the medium. This co-production strategy will help a potential bioprocess of amino acid production with various terpenes.

Lab automation has facilitated synthetic biology applications in an automated workflow, and biofoundry facilities have enabled automated high-throughput experiments of gene cloning and genome engineering to be conducted following a precise experimental design and protocol. However, before-experiment procedures in biofoundry applications have been underdetermined. We aimed to develop a Python-based planning-assistant software, namely Biofoundry Palette, for liquid handler-based experimentation and operation in the biofoundry workflow. Depending on the synthetic biology project, variable information and content information may vary; the Biofoundry Palette provides precise information for the before-experiment units for each process module in the biofoundry workflow. As a demonstration, more than 200 unique information sets, generated by Biofoundry Palette, were used in automated gene cloning or pathway construction. The information on planning and management can potentially help the operator faithfully execute the biofoundry workflow after securing the before-experiment unit, thereby lowering the risk of human errors and performing successful biofoundry operations for synthetic biology applications.

Controlling translational elongation is essential for efficient protein synthesis. Ribosome profiling has revealed that the speed of ribosome movement is correlated with translational efficiency in the translational elongation ramp. In this work, we present a new deep learning model, called DeepTESR, to predict the degree of translational elongation short ramp (TESR) from mRNA sequence. The proposed deep learning model exhibited superior performance in predicting the TESR scores for 226 981 TESR sequences, resulting in the mean absolute error (MAE) of 0.285 and a coefficient of determination R 2 of 0.627, superior to the conventional machine learning models (e.g., MAE of 0.335 and R 2 of 0.571 for LightGBM). We experimentally validated that heterologous fluorescence expression of proteins with randomly selected TESR was moderately correlated with the predictions. Furthermore, a genome-wide analysis of TESR prediction in the 4305 coding sequences of Escherichia coli showed conserved TESRs over the clusters of orthologous groups. In this sense, DeepTESR can be used to predict the degree of TESR for gene expression control and to decipher the mechanism of translational control with ribosome profiling. DeepTESR is available at https://github.com/fmblab/DeepTESR.

Photodynamic therapy (PDT) offers several advantages for treating cancers, but its efficacy is highly dependent on light delivery to activate a photosensitizer. Advances in wireless technologies enable remote delivery of light to tumors, but suffer from key limitations, including low levels of tissue penetration and photosensitizer activation. Here, we introduce DeepLabCut (DLC)-informed low-power wireless telemetry with an integrated thermal/light simulation platform that overcomes the above constraints. The simulator produces an optimized combination of wavelengths and light sources, and DLC-assisted wireless telemetry uses the parameters from the simulator to enable adequate illumination of tumors through high-throughput (<20 mice) and multi-wavelength operation. Together, they establish a range of guidelines for effective PDT regimen design. In vivo Hypericin and Foscan mediated PDT, using cancer xenograft models, demonstrates substantial suppression of tumor growth, warranting further investigation in research and/or clinical settings., (© 2022. The Author(s).)

Objective: Neutralizing antibodies are among the factors used to measure an individual's immune status for the control of infectious diseases. We aimed to confirm the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody levels in patients who had recovered from coronavirus disease 2019 (COVID-19)., Methods: Plasma donors in South Korea who had completely recovered from SARS-CoV-2 infection had follow-up testing to determine the persistence of neutralizing antibodies using a plaque-reduction neutralization test and ELISA., Results: Of the 111 participants-aged 20-29 years, 37/111 (33.3%); 30-39 years, 17/111 (15.3%); 40-49 years, 23/111 (20.7%); 50-59 years, 21/111 (18.9%); 60-65 years, 13/111 (11.7%); male, 43/111 (38.7%); female, 68/111 (61.3%)-66.1% still had neutralizing antibodies approximately 9 months (range 255-302 days) after confirmation of the diagnosis., Conclusions: In this study we analysed the titre of neutralizing antibodies associated with predicting immune status in individuals with natural infection. Information about the persistence and change in levels of neutralizing antibodies against SARS-CoV-2 can be utilized to provide evidence for developing vaccination schedules for individuals with previous infection., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Efficient and versatile DNA assembly frameworks have had an impact on promoting synthetic biology to build complex biological systems. To accelerate system development, laboratory automation (or biofoundry) provides an opportunity to construct organisms and DNA assemblies via computer-aided design. However, a modular cloning (MoClo) system for multiple DNA assemblies limits the biofoundry workflow in terms of simplicity and feasibility by preparing the number of cloning materials such as destination vectors prior to the automation process. Herein, we propose robot-assisted MoClo (RoboMoClo) to accelerate a synthetic biology project with multiple gene expressions at the biofoundry. The architecture of the RoboMoClo framework provides a hybrid strategy of hierarchical gene assembly and iterative gene assembly, and fewer destination vectors compared with other MoClo systems. An industrial bacterium, Corynebacterium glutamicum , was used as a model host for RoboMoClo. After building a biopart library (promoter and terminator; level 0) and evaluating its features (level 1), various transcriptional directions in multiple gene assemblies (level 2) were studied using the RoboMoClo vectors. Among the constructs, the convergent construct exhibited potential transcriptional interference through the collision of RNA polymerases. To study design of experiment-guided lycopene biosynthesis in C. glutamicum (levels 1, 2, and 3), the biofoundry-assisted multiple gene assembly was demonstrated as a proof-of-concept by constructing various sub-pathway units (level 2) and pathway units (level 3) for C. glutamicum . The RoboMoClo framework provides an improved MoClo toolkit for laboratory automation in a synthetic biology application.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus, responsible for outbreaks of a severe respiratory illness in humans with a fatality rate of 30%. Currently, there are no vaccines or United States food and drug administration (FDA)-approved therapeutics for humans. The spike protein displayed on the surface of MERS-CoV functions in the attachment and fusion of virions to host cellular membranes and is the target of the host antibody response. Here, we provide a molecular method for neutralizing MERS-CoV through potent antibody-mediated targeting of the receptor-binding subdomain (RBD) of the spike protein. The structural characterization of the neutralizing antibody (KNIH90-F1) complexed with RBD using X-ray crystallography revealed three critical epitopes (D509, R511, and E513) in the RBD region of the spike protein. Further investigation of MERS-CoV mutants that escaped neutralization by the antibody supported the identification of these epitopes in the RBD region. The neutralizing activity of this antibody is solely provided by these specific molecular structures. This work should contribute to the development of vaccines or therapeutic antibodies for MERS-CoV., (© 2022. The Author(s).)

Motivation: Alignment of protein-protein interaction networks can be used for the unsupervised prediction of functional modules, such as protein complexes and signaling pathways, that are conserved across different species. To date, various algorithms have been proposed for biological network alignment, many of which attempt to incorporate topological similarity between the networks into the alignment process with the goal of constructing accurate and biologically meaningful alignments. Especially, random walk models have been shown to be effective for quantifying the global topological relatedness between nodes that belong to different networks by diffusing node-level similarity along the interaction edges. However, these schemes are not ideal for capturing the local topological similarity between nodes., Results: In this article, we propose MONACO, a novel and versatile network alignment algorithm that finds highly accurate pairwise and multiple network alignments through the iterative optimal matching of 'local' neighborhoods around focal nodes. Extensive performance assessment based on real networks as well as synthetic networks, for which the ground truth is known, demonstrates that MONACO clearly and consistently outperforms all other state-of-the-art network alignment algorithms that we have tested, in terms of accuracy, coherence and topological quality of the aligned network regions. Furthermore, despite the sharply enhanced alignment accuracy, MONACO remains computationally efficient and it scales well with increasing size and number of networks., Availability and Implementation: Matlab implementation is freely available at https://github.com/bjyoontamu/MONACO., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tubulointerstitial abnormalities are predictive of the progression of diabetic kidney disease (DKD), and their targeting may be an effective means for prevention. Proximal tubular (PT) expression of kidney injury molecule (KIM)-1, as well as blood and urinary levels, are increased early in human diabetes and can predict the rate of disease progression. Here, we report that KIM-1 mediates PT uptake of palmitic acid (PA)-bound albumin, leading to enhanced tubule injury with DNA damage, PT cell-cycle arrest, interstitial inflammation and fibrosis, and secondary glomerulosclerosis. Such injury can be ameliorated by genetic ablation of the KIM-1 mucin domain in a high-fat-fed streptozotocin mouse model of DKD. We also identified TW-37 as a small molecule inhibitor of KIM-1-mediated PA-albumin uptake and showed in vivo in a kidney injury model in mice that it ameliorates renal inflammation and fibrosis. Together, our findings support KIM-1 as a new therapeutic target for DKD., Competing Interests: Declaration of interests J.V.B. and T.I. are co-inventors on KIM-1 patents assigned to Mass General Brigham. J.V.B. and A.K.A. have filed a patent for the discovery of TW-37 as inhibitor of KIM-1 to alleviate chronic kidney disease. J.V.B. is a consultant to Cadent, Praxis, Seattle Genetics, Aldeyra, Sarepta, Praxis, and Angion and owns equity in Goldfinch, Innoviva, MediBeacon, DxNow, Verinano, Autonomous Medical Devices, Renalytix, Pacific Biosciences, and Sentien. V.K.K. has an ownership interest in Tizona Therapeutics, Celsius Therapeutics, and Bicara Therapeutics. V.K.K. has financial interests in Biocon Biologic, BioLegend, Elpiscience Biopharmaceutical Ltd., Equilium Inc., and Syngene Intl. V.K.K. is a member of SABs for Elpiscience Biopharmaceutical Ltd., GSK, Rubius Therapeutics, and Tizona Therapeutics. J.V.B.’s and V.K.K.’s interests were reviewed and are managed by Brigham and Women’s Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. S.X. is an employee and shareholder of Celsius Therapeutics. J.H. is a full-time employee of Boehringer Ingelheim Pharmaceuticals, Inc. TW-37 was identified as a KIM-1 inhibitor in a screen supported by Boehringer Ingelheim Pharmaceuticals, Inc., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Industrial application of cyanobacterial poly-β-hydroxybutyrate (PHB) production from CO 2 is currently challenged by slow growth rate and low photoautotrophic PHB productivity of existing cyanobacteria species. Herein, a novel PHB-producing cyanobacterial strain was developed by harnessing fast-growing cyanobacteria Synechococcus elongatus UTEX 2973 with introduction of heterologous phaCAB genes. Under photoautotrophic condition, the engineered strain produced 420 mg L -1 (16.7% of dry cell weight) with the highest specific productivity of 75.2 mg L -1 d -1 . When compared with a native PHB producer Synechocystis PCC 6803 under nitrogen deprivation, the engineered strain exhibited 2.4-fold higher PHB productivity. The performance of the engineered strain was further demonstrated in large scale cultivation using photobioreactor and outdoor cultivation employing industrial flue gas as the sole carbon source. This study can provide a promising solution to address petroleum-based plastic waste and contribute to CO 2 mitigation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In tissue engineering, design of biomaterial with a micro/nano structure is an essential step to mimic extracellular matrix (ECM) and to enhance biomineralization as well as cell biocompatibility. Composite polymeric nanofiber with iron particles/ions has an important role in biomineralization and collagen synthesis for bone tissue engineering. Herein, we report development of polymeric cellulose acetate (CA) nanofibers (17 wt.%) and traces of iron acetates salt (0.5 wt.%) within a polymeric solution to form electrospinning nanofibers mats with iron nanoparticles for bone tissue engineering applications. The resulting mats were characterized using field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), Fourier transform infrared (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The resulted morphology indicated that the average diameter of CA decreased after addition of iron from (395 ± 30) to (266 ± 19) nm and had dense fiber distributions that match those of native ECM. Moreover, addition of iron acetate to CA solution resulted in mats that are thermally stable. The initial decomposition temperature was 300 °C of CA/Fe mat > 270 °C of pure CA. Furthermore, a superior apatite formation resulted in a biomineralization test after 3 days of immersion in stimulated environmental condition. In vitro cell culture experiments demonstrated that the CA/Fe mat was biocompatible to human fetal-osteoblast cells (hFOB) with the ability to support the cell attachment and proliferation. These findings suggest that doping traces of iron acetate has a promising role in composite mats designed for bone tissue engineering as simple and economically nanoscale materials. Furthermore, these biomaterials can be used in a potential future application such as drug delivery, cancer treatment, and antibacterial materials.

Biocontainment systems have been developed to mitigate the concerns regarding biosafety and environmental risk because of the possible escape of genetically modified organisms into the environment following large-scale outdoor cultivation. Here, we present a biocontainment system entailing genetically modified Synechococcus elongatus PCC 7942, also engineered for α-farnesene production using a de-evolutionary strategy. In this approach, the gene cluster encoding the β-carboxysome and the associated carbon concentrating mechanism (CCM) were deleted in the α-farnesene-producing cyanobacteria, resulting in no cell growth and no α-farnesene production at ambient CO 2 concentrations (100% air bubbling). However, cell growth and α-farnesene production were detected in the CCM-deficient strains at high CO 2 concentrations (5% CO 2 [v/v], 10% CO 2 [v/v]), albeit at levels lower than those of the parental control. To overcome this limitation, the overexpression of carbonic anhydrase and bicarbonate transporter genes in the CCM-deficient strains restored cell growth and the production level of α-farnesene (5.0 ± 0.6 mg/L) to that of the parental control. The production of α-farnesene in the later strains strictly depended on CO 2 concentration in the photobioreactor and did not rely on a chemical induction process. Thus, next generation bio-solar cell factories could be promoted with the suggested biocontainment system.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Liver tissue engineering offers a promising strategy for liver failure patients. Since transplantation rejection resulting in vessel thrombosis is regarded as a major hurdle, vascular reconstruction is one of indispensable requirements of whole organ engineering. Here we demonstrated a novel strategy for reconstruction of a vascularized bioengineered human liver (VBHL) using decellularized liver scaffolds in an efficient manner. First we achieved fully functional endothelial coverage of scaffolds by adopting the anti-CD31 aptamer as a potent coating agent for re-endothelialization. Through an ex vivo human blood perfusion that recapitulates the blood coagulation response in humans, we demonstrated significantly reduced platelet aggregation in anti-CD31 aptamer coated scaffolds. We then produced VBHL constructs using liver parenchymal cells and nonparenchymal cells, properly organized into liver-like structures with an aligned vasculature. Interestingly, VBHL constructs displayed prominently enhanced long-term liver-specific functions that are affected by vascular functionality. The VBHL constructs formed perfusable vessel networks in vivo as evidenced by the direct vascular connection between the VBHL constructs and the renal circulation. Furthermore, heterotopic transplantation of VBHL constructs supported liver functions in a rat model of liver fibrosis. Overall, we proposed a new strategy to generate transplantable bioengineered livers characterized by highly functional vascular reconstruction., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To enable circadian control of gene expression in cyanobacteria, we constructed a genetic logic gate (NAND) using orthogonal promoters via modular CRISPR interference. The NAND gates were tested in Synechococcus elongatus PCC 7942 using a fluorescent reporter. The NAND gate dynamics were characterized based on the affinity of the dCas9 complex to the output element. Upon connecting tight gene repressions with the circadian promoter (the purF gene; peak expression at dawn), inversed peak expressions were obtained as an output of the NAND gate although the retroactivities were shown in the ON and OFF states. A dark-responsive genetic element of the NAND gate was also expanded to an AND gate in S. elongatus PCC 7942. These cyanobacterial NAND and AND gates could facilitate the control of gene expressions in dynamic metabolic engineering technologies, thereby enabling the cyanobacteria to serve as biosolar cell factories.

Acute liver failure (ALF) occurs due to severe liver damage that triggers rapid loss of normal liver function. Here, we investigate the usefulness of an injectable liver extracellular matrix (LECM)-rich hydrogel generated from an optimized decellularization protocol incorporated with silver nanoparticles (AgNPs) as a promising therapy for ALF. First, we optimized a non-destructive protocol for rat liver decellularization to obtain ECM-rich well-preserved scaffold. Then, LECM hydrogel generated from two commonly used decellularization protocols were compared by LECM hydrogel obtained from our optimized protocol. The ALF model was induced by an intraperitoneal (IP) thioacetamide (TAA) injection followed by the IP injection of LECM hydrogel, collagen-AgNP mixture, or LECM hydrogel-AgNP mixture. LECM-rich scaffold and hydrogel were successfully obtained using our optimized decellularization protocol. Use of the LECM hydrogel-AgNP mixture to treat TAA-induced ALF greatly improved liver injury and histological liver regeneration. Interleukin-6 and transforming growth factor-beta expressions were significantly reduced, while albumin, hepatocyte growth factor, and Ki67-positive cells were highly expressed. Moreover, aspartate transaminase and alanine transaminase plasma levels and liver homogenate nitric oxide level were significantly lowered. In conclusion, the LECM hydrogel-AgNP mixture has potential efficient therapeutic and regenerative effects on TAA-induced liver injury., (© 2020 Wiley Periodicals LLC.)

To supply the sustainable calorie-free sweetener stevioside, synthetic photosynthetic bacteria were developed to produce ent -kaurenoic acid as a precursor of stevioside directly from CO 2 . By the use of a combinatorial and modular approach for gene expression, including a cytochrome P450 and the corresponding reductase, engineered Synechoccous elongatus PCC 7942 as a model cyanobacterium enabled the biosynthesis of ent -kaurenoic acid at 2.9 ± 0.01 mg L -1 from CO 2 . We found that the order of genes for expression was critical, producing ent -kaurenoic acid by balancing gene expressions and accumulation of the toxic intermediate in a cell. The engineered bacteria allowed the complete biosynthesis of ent -kaurenoic acid, and it will be used for stevioside biosynthesis from CO 2 as a controlled fermentation.

Various incurable eye diseases in companion animals often result in phthisis bulbi and eye removal surgery. Currently, the evisceration method using silicone balls is useful in animals; however, it is not available to those with impaired cornea or severe ocular atrophy. Moreover, ocular implant and prostheses are not widely used because of the diversity in animal size and eye shape, and high manufacturing cost. Here, we produced low-cost and customized artificial eyes, including implant and prosthesis, using computer-aided design and three-dimensional (3D) printing technique. For 3D modeling, the size of the artificial eyes was optimized using B-mode ultrasonography. The design was exported to STL files, and then printed using polycaprolactone (PCL) for prosthesis and mixture of PCL and hydroxyapatite (HA) for ocular implant. The 3D printed artificial eyes could be produced in less than one and half hour. The prosthesis was painted using oil colors and biocompatible resin. Two types of eye removal surgery, including evisceration and enucleation, were performed using two beagle dogs, as a preliminary study. After the surgery, the dogs were clinically evaluated for 6 months and then histopathological evaluation of the implant was done. Ocular implant was biocompatible and host tissue ingrowth was induced after in vivo application. The custom-made prosthesis was cosmetically excellent. Although long-term clinical follow-up might be required, the use of 3D printed-customized artificial eyes may be beneficial for animals that need personalized artificial eye surgery., Competing Interests: The authors have declared that no competing interests exist.

Liver fibrosis results from excessive accumulation of extracellular matrix (ECM) proteins that distort the hepatic architecture. Progression of liver fibrosis results in cirrhosis and liver failure, and often, liver transplantation is required. The decellularized liver tissue contains different components that mimic the natural hepatic environment. We hypothesized that a decellularized liver hydrogel can be used to replace the necrotic hepatocytes and damaged ECM. Therefore, our aim in this study is to develop a therapy for treating liver fibrosis. Mice livers were decellularized and processed to form a hepatic hydrogel. We evaluated the biocompatibility and bioactivity of the hydrogel. The ability of the hydrogel to enhance the migration of hepatocytes and endothelial cells was investigated. Human hepatic stellate cell line (LX-2) activated by transforming growth factor-β1 (TGF-β1) was used as in vitro model for fibrogenesis. Then, the hydrogel was injected into the liver parenchyma of mice after the induction of liver fibrosis using thioacetamide. The resulting hydrogel maintained a complex composition, which included glycosaminoglycans, collagen, elastin, and growth factors. Hepatocytes and endothelial cells were shown to migrate toward the hydrogel in vitro. Liver hydrogel improved TGF-β1-induced LX-2 cells activation via blocking the TGF-β1/Smad pathway. The matrix was delivered successfully in vivo and enhanced the reduction of fibrosis and recovery to a nearly normal structure. In conclusion, we have demonstrated that the liver hydrogel can be utilized as an injectable biomaterial for liver tissue engineering in order to reduce the degree of fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Due to their very poor proliferative capacity, the dysfunction of corneal endothelial cells can sometimes lead to incurable eye diseases that require corneal transplantation. Although many studies have been performed to reconstruct corneal endothelial cells, corneal transplantation is still considered to be the established approach. In this study, we developed bio-engineered Descemet stripping endothelial (DSE) layers, using porcine cornea and induced pluripotent stem cell (iPSC)-derived corneal endothelial cells (iCECs). First, we optimized a protocol to prepare an ultra-thin and decellularized Descemet stripping (DS) scaffold from porcine cornea. Our DS layers show over 90% transparency compared to the control. Porcine-derived cells and xenogenic antigens disappeared, whereas the collagen matrix remained in the graft. Next, corneal endothelial cell lines or iCECs were seeded on the decellularized DS graft and cultured for 7 days. The drying method reduced graft rolling and edema, and increased transparency during culture. The reseeded cells were evenly distributed over the graft, and most of the cells survived. Although future clinical studies are warranted, engineered DSE tissues using xenogenic tissues and stem cells will be useful tools for the treatment of incurable corneal diseases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The generation of a transplantable liver scaffold is crucial for the treatment of end-stage liver failure. Unfortunately, decellularized liver scaffolds suffer from lack of bioactive molecules and functionality. In this study, we conjugated homogenized liver-extracellular matrix (ECM) into a decellularized liver in a rat model to improve its structural and functional properties. The homogenized ECM was prepared, characterized, and subsequently perfused into ethyl carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) activated liver scaffolds. Various techniques were performed to confirm the improvements that were accomplished through the conjugation process; these included micro/ultra-structural analyses, biochemical analysis of ECM components, DNA quantification, swelling ratio, structural stability, calcification properties, platelet activation study, static and dynamic seeding with EAhy926 endothelial cells and HepG2 hepatocarcinoma cells, subcutaneous implantation and intrahepatic transplantation. The results showed that the conjugated scaffolds have superior micro- and ultrastructural and biochemical characteristics. In addition, DNA contents, swelling ratios, calcification properties, platelet reactions, and host inflammatory reactions were not altered with the conjugation process. The conjugated scaffolds revealed better cellular spreading and popularity compared to the non-conjugated scaffolds. Intrahepatic transplantation showed that the conjugated scaffold had higher popularity of hepatic regenerative cells with better angiogenesis. The conjugation of the decellularized liver scaffold with homogenized liver-ECM is a promising tool to improve the quality of the generated scaffold for further transplantation., (© 2020 Wiley Periodicals, Inc.)

Cyanobacteria are photosynthetic microorganisms that are capable of converting CO 2 to value-added chemicals. Engineering of cyanobacteria with synthetic biology tools, including the CRISPR-Cas system, has allowed an opportunity for biological CO 2 utilization. Here, we described natural CRISPR-Cas systems for understanding cyanobacterial genomics and synthetic CRISPR-Cas systems for metabolic engineering applications. The natural CRISPR-Cas systems in cyanobacteria have been identified as Class 1, with type I and III, and some Class 2, with type V, as an adaptive immune system against viral invasion. As synthetic tools, CRISPR-Cas9 and -Cas12a have been successfully established in cyanobacteria to delete a target gene without a selection marker. Deactivated Cas9 and Cas12a have also been used to repress genes for metabolic engineering. In addition, a perspective on how advanced CRISPR-Cas systems and a pool of the guide RNAs can be advantageous for precise genome engineering and understanding of unknown functions was discussed for advanced engineering of cyanobacteria., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A systematic and combinatorial optimization has been employed to metabolically engineer microbes for identifying key gene targets for overexpression to increase the intermediate pools for terpenoid production. Herein, the methylerythritol 4-phosphate (MEP) pathway in Corynebacterium glutamicum , an industrial host, was investigated to identify the key genes whose overexpression would improve the production of farnesyl diphosphate (FPP)-derived terpenoids (squalene and α-farnesene). Using a combinatorial approach with the single, double, and triple expression of genes in the MEP pathway in a high-throughput fermentation, overexpression of the ispDF genes, along with the known dxs and idi genes, was most effective at increasing the squalene contents, i.e., by 14-fold. The dxr gene was identified as the key target enzyme for α-farnesene production. This result could provide fundamental information for improving the metabolic engineering of C. glutamicum for terpene production via an optimized MEP pathway.

In cyanobacteria, metabolic engineering using synthetic biology tools is limited to build a biosolar cell factory that converts CO 2 to value-added chemicals, as repression of essential genes has not been achieved. In this study, we developed a dCas12a-mediated CRISPR interference system (CRISPRi-dCas12a) in cyanobacteria that effectively blocked the transcriptional initiation by means of a CRISPR-RNA (crRNA) and 19-nt direct repeat, resulting in 53-94% gene repression. The repression of multiple genes in a single crRNA array was also successfully achieved without a loss in repression strength. In addition, as a demonstration of the dCas12a-mediated CRISPRi for metabolic engineering, photosynthetic squalene production was improved by repressing the essential genes of either acnB encoding for aconitase or cpcB 2 encoding for phycocyanin β-subunit in Synechococcus elongatus PCC 7942. The ability to regulate gene repression will promote the construction of biosolar cell factories to produce value-added chemicals.

Economically feasible photosynthetic cultivation of microalgal and cyanobacterial strains is crucial for the biological conversion of CO 2 and potential CO 2 mitigation to challenge global warming. To overcome the economic barriers, the production of value-added chemicals was desired by compensating for the overall processing cost. Here, we engineered cyanobacteria for photosynthetic squalene production and cultivated them in a scalable photobioreactor using industrial flue gas. First, an inducer-free gene expression system was developed for the cyanobacteria to lower production const. Then, the recombinant cyanobacteria were cultivated in a closed photobioreactor (100 L) using flue gas (5% CO 2 ) as the sole carbon source under natural sunlight as the only energy source. Seasonal light intensities and temperatures were analyzed along with cyanobacterial cell growth and squalene production in August and October 2019. As a result, the effective irradiation hours were the most critical factor for the large-scale cultivation of cyanobacteria. Thus, an automated photobioprocess system will be developed based on the regional light sources.

Designing synthetic pathways for efficient CO 2 fixation and conversion is essential for sustainable chemical production. Here we have designed a synthetic acetate-acetyl-CoA/malonyl-CoA (AAM) bypass to overcome an enzymatic activity of pyruvate dehydrogenase complex. This synthetic pathway utilizes acetate assimilation and carbon rearrangements using a methyl malonyl-CoA carboxyltransferase. We demonstrated direct conversion of CO 2 into acetyl-CoA-derived acetone as an example in photosynthetic Synechococcus elongatus PCC 7942 by increasing the acetyl-CoA pools. The engineered cyanobacterial strain with the AAM-bypass produced 0.41 g/L of acetone at 0.71 m/day of molar productivity. This work clearly shows that the synthetic pyruvate dehydrogenase bypass (AAM-bypass) is a key factor for the high-level production of an acetyl-CoA-derived chemical in photosynthetic organisms., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Auricular cartilage reconstruction represents one of the greatest challenges for otolaryngology-head and neck surgery. The native structure and composition of the auricular cartilage can be achieved by combining a suitable chondrogenic cell source with an appropriate scaffold. In reconstructive surgery for cartilage tissue, autogenous cartilage is considered to be the best chondrogenic cell source. Polycaprolactone is mainly used as a tissue-engineered scaffold owing to its mechanical properties, miscibility with a large range of other polymers, and biodegradability. In this study, scaffolds with or without autogenous minced auricular cartilage were implanted bilaterally in rabbits for auricular regeneration. Six weeks (n = 4) and 16 weeks (n = 4) after implantation, real-time quantitative reverse transcription polymerase chain reaction and histology were used to assess the regeneration of the auricular cartilage. Quantitative reverse transcription polymerase chain reaction analysis revealed that the messenger RNA expression of aggrecan, collagen I, and collagen II was higher in scaffolds with 50% minced cartilage than the scaffold-only groups or scaffolds with 30% minced cartilage (P < 0.05). Furthermore, histological analysis demonstrated significantly superior cartilage regeneration in scaffolds with the minced cartilage group compared with the scaffold-only and control groups (P < 0.05). Autogenous cartilage can be easily obtained and loaded onto a scaffold to promote the presence of chondrogenic cells, allowing for an improvement of the reconstruction of auricular cartilage. Here, the regeneration of auricular cartilage was also successful in the 50% minced cartilage group. The results presented in this study could have clinical implications, as they demonstrate the potential of a 1-stage process for auricular reconstruction.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infection and continues to infect humans, thereby contributing to a high mortality rate (34.3% in 2019). In the absence of an available licensed vaccine and antiviral agent, therapeutic human antibodies have been suggested as candidates for treatment. In this study, human monoclonal antibodies were isolated by sorting B cells from patient's PBMC cells with prefusion stabilized spike (S) probes and a direct immunoglobulin cloning strategy. We identified six receptor-binding domain (RBD)-specific and five S1 (non-RBD)-specific antibodies, among which, only the RBD-specific antibodies showed high neutralizing potency (IC50 0.006-1.787 μg/ml) as well as high affinity to RBD. Notably, passive immunization using a highly potent antibody (KNIH90-F1) at a relatively low dose (2 mg/kg) completely protected transgenic mice expressing human DPP4 against MERS-CoV lethal challenge. These results suggested that human monoclonal antibodies isolated by using the rationally designed prefusion MERS-CoV S probe could be considered potential candidates for the development of therapeutic and/or prophylactic antiviral agents for MERS-CoV human infection., Competing Interests: We also state that competing interests: the patent name “MONOCLONAL ANTIBODY FOR SPIKE PROTEIN OF MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS, AND USE THEREOF” and No. 10-1895229 (Korean patent) (PCT application No. PCT KR2018009754). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Agrimonia pilosa (AP) and Rhus gall (RG) are traditional medicinal plants. The bioflavonoid composition standardized by HPLC analysis was named APRG64. Despite many studies reported to beneficial bioactivities of AP and RG, very limited range of toxicity tests have documented. So, we did experiment diversely on the toxicity tests of the substance APRG64. Genotoxicity (mammalian chromosomal aberration test, micronoucleus test) against APRG64, acute and sub-chronic toxicity test from rodent/non-rodent, and systemic safety pharmacology test were conducted. As a result of the test, genotoxicity against APRG64 was not observed. The NOAEL of rodents was confirmed as 2000 mg/kg/day and non-rodents was confirmed as 500 mg/kg/day. In addition, systemic safety pharmacological toxicity (effects on respiratory system, central nervous system, cardiovascular system) following administration of APRG64 was not observed. Finally, we accomplished ten potential toxicity tests and evaluated extensive safety of APRG64. Consequently, APRG64 may be a promising material for nutraceuticals and natural medicines., Competing Interests: Conflict of interestThere are no conflicts to declare., (© Korean Society of Toxicology 2020.)

Background/aim: Bone tissue engineering is an emerging field of regenerative medicine that holds promise for the restoration of bones affected by trauma, neoplastic diseases, and congenital deformity. During the past decade, bone tissue engineering has evolved from the use of biomaterials that can only replace small areas of damaged bone, to the use of scaffolds in which grafts can be seeded before implantation. This case report proposes an alternative option for a veterinary patient suffering from ectrodactyly, which is one of several congenital deformities in dogs. A 2-month-old male toy poodle dog with ectrodactyly was treated using several stages of surgery involving pancarpal arthrodesis, limb lengthening, and bone tissue engineering techniques., Results and Conclusion: Over a period of 2 years, the operated limb gained almost the same function as the contralateral limb. Bone tissue engineering techniques can be used for the treatment of congenital deformities in dogs., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Although the generation of ETV2-induced endothelial cells (iECs) from human fibroblasts serves as a novel therapeutic strategy in regenerative medicine, the process is inefficient, resulting in incomplete iEC angiogenesis. Therefore, we employed chromatin immunoprecipitation (ChIP) sequencing and identified molecular mechanisms underlying ETV2-mediated endothelial transdifferentiation to efficiently produce iECs retaining appropriate functionality in long-term culture. We revealed that the majority of ETV2 targets in human fibroblasts are related to vasculature development and signaling transduction pathways, including Rap1 signaling. From a screening of signaling pathway modulators, we confirmed that forskolin facilitated efficient and rapid iEC reprogramming via activation of the cyclic AMP (cAMP)/exchange proteins directly activated by cAMP (EPAC)/RAP1 axis. The iECs obtained via cAMP signaling activation showed superior angiogenesis in vivo as well as in vitro. Moreover, these cells could form aligned endothelium along the vascular lumen ex vivo when seeded into decellularized liver scaffold. Overall, our study provided evidence that the cAMP/EPAC/RAP1 axis is required for the efficient generation of iECs with angiogenesis potential., (Copyright © 2019. Published by Elsevier Inc.)

Bioengineering a functional organ holds great potential to overcome the current gap between the organ need and shortage of available organs. Whole organ decellularization allows the removal of cells from large-scale organs, leaving behind extracellular matrices containing different growth factors, structural proteins, and a vascular network with a bare surface. Successful application of decellularized tissues as transplantable organs is hampered by the inability to completely reline the vasculature by endothelial cells (ECs), leading to blood coagulation, loss of vascular patency, and subsequent death of reseeded cells. Therefore, an intact, continuous layer of endothelium is essential to maintain proper functioning of the vascular system, which includes the transfer of nutrients to surrounding tissues and protecting other types of cells from shear stress. Here, we aimed to summarize the available cell sources that can be used for reendothelialization in addition to different trials performed by researchers to reconstruct vascularization of decellularized solid organs. Additionally, different techniques for enhancing reendothelialization and the methods used for evaluating reendothelialization efficiency along with the future prospective applications of this field are discussed. STATEMENT OF SIGNIFICANCE: Despite the great progress in whole organ decellularization, reconstruction of vasculature within the engineered constructs is still a major roadblock. Reconstructed endothelium acts as a multifunctional barrier of vessels, which can reduce thrombosis and help delivering of oxygen and nutrients throughout the whole organ. Successful reendothelialization can be achieved through reseeding of appropriate cell types on the naked vasculature with or without modification of its surface. Here, we present the current research milestones that so far established to reconstruct the vascular network in addition to the methods used for evaluating the efficiency of reendotheilization. Thus, this review is quite significant and will aid the researchers to know where we stand toward biofabricating a transplantable organ from decellularizd extracellular matrix., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Comparative network analysis provides effective computational means for gaining novel insights into the structural and functional compositions of biological networks. In recent years, various methods have been developed for biological network alignment, whose main goal is to identify important similarities and critical differences between networks in terms of their topology and composition. A major impediment to advancing network alignment techniques has been the lack of gold-standard benchmarks that can be used for accurate and comprehensive performance assessment of such algorithms. The original NAPAbench (network alignment performance assessment benchmark) was developed to address this problem, and it has been widely utilized by many researchers for the development, evaluation, and comparison of novel network alignment techniques. In this work, we introduce NAPAbench 2-a major update of the original NAPAbench that was introduced in 2012. NAPAbench 2 includes a completely redesigned network synthesis algorithm that can generate protein-protein interaction (PPI) network families whose characteristics closely match those of the latest real PPI networks. Furthermore, the network synthesis algorithm comes with an intuitive GUI that allows users to easily generate PPI network families with an arbitrary number of networks of any size, according to a flexible user-defined phylogeny. In addition, NAPAbench 2 provides updated benchmark datasets-created using the redesigned network synthesis algorithm-which can be used for comprehensive performance assessment of network alignment algorithms and their scalability., Competing Interests: The authors have declared that no competing interests exist.